L’apurinic/apyrimidic endonuclease 1 (APE1) est une protéine multifonctionnelle qui joue un rôle important dans la voie de réparation de l’ADN par excision de base. Elle sert également de coactivateur de transcription et est aussi impliquée dans le métabolisme de l’ARN et la régulation redox. APE1 peut cliver les sites AP ainsi que retirer des groupements, sur des extrémités 3’ créées suite à des bris simple brin, qui bloquent les autres enzymes de réparation, permettant de poursuivre la réparation de l’ADN, puisqu’elle possède plusieurs activités de réparation de l’ADN comme une activité phosphodiestérase 3’ et une activité exonucléase 3’→5’. Les cellules de mammifères ayant subi un knockdown d’APE1 présentent une grande sensibilité face à de nombreux agents génotoxiques. APE1 ne possède qu’une seule cystéine située au 65e acide aminé. Celle-ci est nécessaire pour maintenir l’état de réduction de nombreux activateurs de transcription tels que p53, NF-κB, AP-1, c-Jun at c-Fos. Ainsi, elle se retrouve impliquée dans la régulation de l’expression génique. APE1 passe également à travers au moins 4 types de modifications post-traductionnelles : l’acétylation, la désacétylation, la phosphorylation et l’ubiquitylation. La façon dont APE1 est recrutée pour accomplir ses différentes fonctions biologiques demeure un mystère, bien que cela puisse être relié à sa capacité d’interaction avec de multiples partenaires différents. Sous des conditions de croissance normales, il a été démontré qu’APE1 interagit avec de nombreux partenaires impliqués dans de multiples fonctions. Nous émettons l’hypothèse que l’état d’oxydation d’APE1 est ce qui contrôle les partenaires avec lesquels la protéine interagira, lui permettant d’accomplir des fonctions précises. Dans cette étude nous démontrons que le peroxyde d’hydrogène altère le réseau d’interactions d’APE1. Un nouveau partenaire d’interaction d’APE1, Prdx1, un membre de la famille des peroxirédoxines responsable de récupérer le peroxyde d’hydrogène, est caractérisé. Nous démontrons qu’un knockdown de Prdx1 n’affecte pas l’activité de réparation de l’ADN d’APE1, mais altère sa détection et sa distribution cellulaire à l’intérieur des cellules HepG2 conduisant à une induction accrue de l’interleukine 8 (IL-8). L’IL8 est une chimiokine impliquée dans le stress cellulaire en conditions physiologiques et en cas de stress oxydatif. Il a été démontré que l’induction de l’IL-8 est dépendante d’APE1 indiquant que Prdx1 pourrait réguler l’activité transcriptionnelle d’APE1. Il a été découvert que Prdx1 est impliquée dans la régulation redox suite à une réponse initiée par le peroxyde d’hydrogène. Ce dernier possède un rôle important comme molécule de signalisation dans de nombreux processus biologiques. Nous montrons que Prdx1 est nécessaire pour réduire APE1 dans le cytoplasme en réponse à la présence de H2O2. En présence de Prdx1, la fraction d’APE1 présent dans le cytoplasme est réduite suite à une exposition au peroxyde d’hydrogène, et Prdx1 est hyperoxydé suite à l’interaction entre les deux molécules. Cela suggère que le signal, que produit le peroxyde d’hydrogène, sur APE1 passe par Prdx1. Un knockdown d’APE1 diminue la conversion de la forme dimérique de Prdx1 vers la forme monomérique. Cette observation implique qu’APE1 pourrait être impliquée dans la régulation de l’activité catalytique de Prdx1 en accélérant son hyperoxydation. / Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein, which play important roles in base excision repair (BER) pathway and serve as transcriptional co-activator. APE1 is also involved in RNA metabolism and redox regulation. APE1 can cleave abasic sites and process 3’-blocking termini into 3’-OH for DNA repair replication as it posseses several DNA repair activities including AP endonuclease, 3’-phosphodiesterase and 3’ to 5’-exonuclease. Mammalian cells knockdown for APE1 are very sensitive to various DNA damaging agents. APE1 has a unique cysteine C65, which is required to maintain the reduced state of several transcriptional activators such as p53, NF-кB, AP-1, c-Jun, and c-Fos and therefore is involved in the regulation of gene expression. APE1 also undergoes at least four types of post-translational modifications that include acetylation, deacetylation, phosphorylation and ubiquitylation. How APE1 is being recruited to execute the various biological functions remains a challenge, although this could be directly related to its ability to interact with multiple different partners. Under normal growth conditions, APE1 has been shown to interact with a number of proteins that are involved in various functions. We propose that the oxidative state of APE1 governs its interacting partners thereby allowing the protein to perform specific functions. In this study we find that APE1 interactome alters in response to hydrogen peroxide. One novel APE1 interacting partner Prdx1, a member of the peroxiredoxin family that can scavenge hydrogen peroxide is characterized. We demonstrate that knockdown of Prdx1 did not impair APE1 DNA repair activity, but alters APE1 detection, and subcellular distribution in HepG2 cells leading to the induction of interleukin 8 (IL-8). IL-8 is a pro-inflammatory chemokine involved in cellular stress, under physiological and iv oxidative stress conditions. It has been shown that the induction of IL-8 is dependent on APE1 indicating Prdx1 may regulate APE1 transcriptional activity. Prdx1 has been discovered to be involved in the redox regulation of cell signaling initiated by hydrogen peroxide, which has important roles as a signaling molecule in the regulation of a variety of biological processes. Prdx1 exists as a dimer in the cells and we show that Prdx1 is required to reduce APE1 in the cytoplasm in response to H2O2. During this process, the dimeric form of Prdx1 is converted to the oxidized monomeric form. Interestingly, the H2O2-induced conversion of Prdx1 to the monomeric form is dependent upon the presence of APE1. These observations imply that there is a tight regulatory network existing between APE1 and Prdx1.

Nous avons récemment démontré que les espèces réactives oxygénées induisent une augmentation de l’expression des protéines Giα dans les cellules du muscle lisse vasculaire (CMLV) provenant d’aortes de rats spontanément hypertendus (SHR, de l’anglais spontaneously hypertensive rats). La présente étude a pour but d’étudier les effets du peroxyde d’hydrogène (H2O2), un oxydant qui induit le stress oxydatif, sur l’expression de Giα et sur l’activité de l’adénylate cyclase, et d’explorer les voies de signalisation sous-jacentes responsables de cette réponse. Nos résultats montrent que H2O2 induit une augmentation de l’expression des protéines Giα-2 et Giα-3 de manière dose- et temps-dépendante avec une augmentation maximale de 40-50% à 100 µM après 1 heure, sans affecter l’expression de Gsα. L’expression des protéines Giα a été maintenue au niveau normal en presence de AG 1478, AG1295, PD98059 et la wortmannine, des inhibiteurs d’EGF-R (de l’anglais epidermal growth factor receptor), PDGFR-β (de l’anglais platelet-derived growth factor receptor β), de la voie de signalisation ras-ERK1/2 (de l’anglais extracellular regulated kinase1/2), et de la voie de la PI3Kinase-AKT (de l’anglais phosphatidyl inositol-3 kinase), respectivement. En outre, le traitement des CMLV avec H2O2 a induit une augmentation du degré de phosphorylation d’EGF-R, PDGF-R, ERK1/2 et AKT; et cette expression a été maintenue au niveau témoin par leurs inhibiteurs respectifs. Les inhibiteurs d’EGF-R et PDGF-R ont aussi induit une diminution du degré de phosphorylation de ERK1/2, et AKT/PKB. En outre, la transfection des cellules avec le siRNA (de l’anglais, small interfering ribonucleic acid) de EGF-R et PDGFR-β a atténué la surexpression des protéines Giα-2 et Giα-3 induite par le traitement au H2O2. La surexpression des protéines Giα induite par H2O2 a été corrélée avec une augmentation de la fonction de la protéine Giα. L’inhibition de l’activité de l’adénylate cyclase par de faibles concentrations de GTPγS après stimulation par la forskoline a augmenté de 20% dans les cellules traitées au H2O2. En outre, le traitement des CMLV au H2O2 a aussi accru l’inhibition de l’activité de l’adénylate cyclase par les hormones inhibitrices telles que l’angiotensine II, oxotrémorine et C-ANP4-23. D’autre part, la stimulation de l’adénylate cyclase induite par GTPγS, glucagon, isoprotérénol, forskoline, et le fluorure de sodium (NaF) a été atténuée de façon significative dans les cellules traitées au H2O2. Ces résultats suggèrent que H2O2 induit la surexpression des protéines Giα-2 and Giα-3 via la transactivation des récepteurs des facteurs de croissance EGF-R, PDGFR-β et l’activation des voies de signalisation ras-ERK1/2 et PI3K-AKT Mot-cles: Protéines Giα, peroxyde d’hydrogène, stress oxydant, récepteurs des facteurs de croissance, MAP kinases, adénylate cyclase, hypertension / We recently have shown that reactive oxygen species contribute to the enhanced expression of Giα proteins in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR). The present study was undertaken to examine if H2O2, an oxidant that induces oxidative stress could also enhance the expression of Giα proteins and associated adenylyl cyclase signalling in aortic VSMC and to further explore the underlying signaling pathways responsible for this response. Treatment of cells with H2O2 increased the expression of Giα-2 and Giα-3 proteins but not that of Gsα proteins in a concentration- and time-dependent manner. A maximal increase of 40-50% was observed at 100µM and 1h. The enhanced expression of Giα proteins was restored to control levels by AG 1478, AG1295, PD98059 and wortmannin, inhibitors of epidermal growth factor receptor (EGF-R), platelet-derived growth factor receptor (PDGFR-β), the mitogen-activated protein kinase (MEK1/2), and PI3 kinase respectively. In addition, treatment of VSMC with H2O2 also increased the phosphorylation of EGF-R, PDGF-R, ERK1/2 and AKT and this increased phosphorylation was attenuated to control levels by the respective inhibitors, whereas the inhibitors of EGF-R and PDGE-R also attenuated the enhanced phosphorylation of ERK1/2 and AKT to control levels. Transfection of cells with EGF-R and PDGFR-β siRNA followed by H2O2 treatment restored the H2O2-induced enhanced expression of Giα-2 and Giα-3 proteins to control levels. The increased expression of Giα proteins by H2O2 was reflected in the increased Gi functions. The inhibition of forskolin (FSK)-stimulated AC activity by low concentration of GTPγS (receptor- independent Gi functions) was increased by about 20% by H2O2 treatment. Moreover, treatment of cells with H2O2 also resulted in an increased Ang II-, C-ANP4-23, and oxotremorine-mediated inhibition of AC (receptor-dependent functions of Gi). On the other hand, Gsα-mediated stimulation of AC by GTPγS, glucagon, isoproterenol, FSK, and NaF was significantly decreased in H2O2-treated cells. These results suggest that H2O2 increases the expression of Giα-2 and Giα-3 proteins in VSMC through the transactivation of EGF-R, PDGFR-β and associated ERK1/2 and PI3K signalling pathways. Keywords: Giα proteins, hydrogen peroxide, adenylyl cyclase, oxidative stress, MAP kinase, growth factor receptors, hypertension.

This thesis describes the catalytic asymmetric epoxidation of olefins mediated by chiral iminum salts. The first chapter introduces some of the most novel and effective catalytic asymmetric methods for preparing chiral oxiranes. The second chapter is divided into three sections. The first section of chapter two is dedicated to our efforts to develop new aqueous oxidative conditions using both hydrogen peroxide and sodium hypochlorite as efficient, green oxidants that remove the temperature boundaries observed with the use of Oxone® as the stoichiometric oxidant. A wider range of available temperatures was examined allowing optimization of both oxidative systems. Ethereal hydrogen peroxide was observed to mediate asymmetric epoxidation within an acetonitrile monophasic co-solvent system giving enantioselectivities of up to 56%. When sodium hypochlorite was used in a biphasic solvent system in conjunction with dichloromethane; it was observed to mediate oxidation of the substrate alkenes in up to 71% ee. The second and third sections of chapter two are dedicated to our efforts to synthesize chiral iminium salts as catalysts for asymmetric epoxidation based on a biphenyl azepinium salt catalyst structure. From previous work within the Page group, the asymmetric synthesis and subsequent defined stereochemistry of a chiral carbon atom α to the iminium nitrogen atom was shown to have significant effect on the enantiocontrol of epoxidation using the iminium salt catalyst. Work was completed on biphenyl azepinium salt catalysts, inserting an alkyl or aryl Grignard reagent into the iminium bond using a pre-defined dioxane unit as a chiral auxiliary. Oxidation of the subsequent azepine gave a single diastereoisomerically pure azepinium salt. The methyl analogue of this sub-family of azepinium catalysts has been shown to give up to 81% ee for epoxidation of 1-phenylcyclohexene, furthermore, the binaphthalene azepinium salt with an additional methyl group was also synthesized and was shown to give up to 93% for epoxidation of 1-phenylcyclohexene. Continuation of the substitution α to the nitrogen atom gave rise to an interesting tetracyclic (biphenyl) azepinum salt catalyst. Construction of an asymmetric oxazolidine ring unit encapsulating the azepinium nitrogen and one of the methylene carbon atoms was achieved. In doing so two chiral centres α to the nitrogen atom were generated. The azepinium chiral carbon atom was populated by an addition methyl group with variation in the substitution on the oxazolidine chiral carbon atom. The benzyl analogue of this sub-family of tetracyclic azepinium catalysts has shown to give up to 79% ee for epoxidation 1-phenylcyclohexene. The third chapter is the experimental section and is dedicated to the methods of synthesis and characterization of the compounds mentioned in the previous chapter. X-ray reports regarding the crystallographic analysis of the structures presented in chapter two are provided in appendix A. Appendix B contains the analytical spectra for the determination of enantiomeric excess of the epoxides.

Dissolution of the UO2 matrix is one of the potential routes for radionuclide release in a future deep geological repository for spent nuclear fuel. This doctoral thesis focuses on interfacial reactions of relevance in radiation-induced dissolution of UO2 and is divided in two parts: In the first part, we sought to explore the effects of solid phase composition: The impact of surface stoichiometry on the reactivity of UO2 towards aqueous radiolytic oxidants was studied. H2O2 reacts substantially faster with stoichiometric UO2 than with hyperstoichiometric UO2. In addition, the release of uranium from stoichiometric UO2 is lower than from hyperstoichiometric UO2. The behavior of stoichiometric powder changes with exposure to H2O2, approaching the behavior of hyperstoichiometric UO2 with the number of consecutive H2O2 additions. The impact of Gd-doping on the oxidative dissolution of UO2 in an aqueous system was investigated. A significant decrease in uranium dissolution and higher stability towards H2O2 for (U,Gd)O2 pellets compared to standard UO2 was found. In the second part, we sought to look at the effect of external factors: The surface reactivity of H2 and O2 was studied to understand the overall oxide surface reactivity of aqueous molecular radiolysis products. The results showed that hydrogen-abstracting radicals and H2O2 are formed in these systems. Identical experiments performed in aqueous systems containing UO2 powder showed that the simultaneous presence of H2 and O2 enhances the oxidative dissolution of UO2 compared to a system not containing H2. The effect of groundwater components such as bentonite and sulfide on the oxidative dissolution of UO2 was also explored. The presence of bentonite and sulfide in water could either delay or prevent in part the release of uranium to the environment. The Pd catalyzed H2 effect is more powerful than the sulfide effect. The poisoning of Pd catalyst is not observed under the conditions studied. / <p>QC 20170421</p>

A mancha-marrom de Alternaria causada pelo fungo Alternaria alternata f.sp. citri patótipo \"tangerina\" é uma das principais doenças que acometem a cultivo de tangerinas (Citrus reticulata) e alguns de seus híbridos. A infecção ocorre principalmente em folhas e frutos jovens que senescem prematuramente. Para realização de estudos com a finalidade de obtenção de cultivares resistentes, ou para a compreensão dos processos de interação planta-patógeno, é necessária a produção massal de esporos in vitro. Este trabalho, no primeiro momento, buscou desenvolver um método eficaz de obtenção de esporos do fungo em meio de cultura. Para isso, comparou os efeitos de diferentes meios de cultura (BDA e CaCO3-A) e tipos de iluminação (LED e NUV). Além do número de esporos, características como germinação, pigmentação, formação de apressórios e patogenicidade foram avaliadas. Descobriu-se que o melhor método de produção de esporos consistiu da manutenção de colônias em meio CaCO3-A por 3 dias sob LED e em seguida por 4 dias sob NUV, com fotoperíodo de 12 h. A partir de então, no segundo momento, realizaram-se testes com técnicas de microscopia e bioquímica fitopatológica para analisar a interação entre o patógeno e folhas das cultivares suscetíveis Ponkan e Murcott e da resistente Fremont. Notou-se por meio de microscopia eletrônica de varredura (MEV) que, independentemente da cultivar, o fungo germinou sobre as folhas, formou apressórios e entrou em cavidades estomáticas. Não foi observada, porém, a colonização de tecidos por meio de microscopia de luz. As cultivares suscetíveis responderam à inoculação com acúmulo de peróxido de hidrogênio e reação de hipersensibilidade. A cultivar resistente, por sua vez, não apresentou reação de hipersensibilidade e aumentou a síntese de enzimas relacionadas ao estresse oxidativo (SOD e CAT) e eliminação de espécies reativas de oxigênio. Como se trata de um fungo necrotrófico capaz de detoxificar espécies reativas de oxigênio, presumiu-se que a reação de hipersensibilidade e a morte celular nas cultivares suscetíveis favorecem a infecção e colonização do patógeno, enquanto que na cultivar resistente a ausência de morte celular e detoxificação de espécies reativas de oxigênio podem estar envolvidas na sua capacidade de defesa. Nas cultivares suscetíveis, verificou-se, por meio de microscopia eletrônica de transmissão (MET), plasmólise e fragmentação de membranas plasmáticas, local indicado como sítio de ação da toxina ACT produzida pelo patógeno. As células da cultivar resistente não apresentaram plasmólise ou alterações nas membranas plasmáticas em decorrência da inoculação do patógeno. / Alternaria brown spot caused by Alternaria alternate f.sp. citri pathotype \"tangerine\" is a important disease that affects tangerine (Citrus reticulate), and some of its hybrids. The infection happens mainly in young leaves and fruits and may cause premature senescence of tissues. To carry out studies in order to obtain resistant cultivars, or to understand the processes of plant-pathogen interaction, it is necessary abundant in vitro spores production. This work, in the first moment, aimed to develop an efficient method for in vitro spore production of the fungus. It was compared the effects of different culture media (BDA and CaCO3-A) and light (LED and NUV). Besides the number of spores, characteristics such as germination, pigmentation, apressoria formation and pathogenicity were investigated. It was found that the best method of spore production consisted of maintaining colonies on CaCO3-A medium for 3 days under LED and then for 4 days under NUV, with photoperiod of 12 h. Thus, in the second moment, assays were carried out using microscopy and phytopathological biochemistry techniques to analyze the interaction between the pathogen and leaves of the susceptible cultivars Ponkan and Murcott, and the resistant Fremont. It was noted by scanning electron microscopy (SEM) that independently of the cultivar, the fungus germinated on the leaves, formed apressoria and grew into stomatal cavities. However, colonization of tissues was not observed by light microscopy. Susceptible cultivars responded to inoculation with accumulation of hydrogen peroxide and hypersensitivity reaction. The resistant cultivar did not show hypersensitivity reaction, but increased the synthesis of enzymes (CAT and SOD) related to oxidative stress and elimination of reactive oxygen species. As a necrotrophic pathogen able to detoxify reactive oxygen species, it was assumed that the hypersensitivity reaction and cell death in the susceptible cultivars contribute to infection and colonization of the pathogen. On the other hand, the absence of cell death and detoxification of reactive oxygen species verified in the resistant cultivar may be involved on its capacity of defense. Cell plamolysis and plasma membranes fragmentation, indicated as the site of action of the ACT toxin produced by the pathogen, were verified in the susceptible cultivars by transmission electron microscopy (TEM). The cells of the resistant cultivar did not show plasmolysis or alterations in the plasma membranes due to pathogen inoculation.

[pt] O tratamento de águas de resfriamento normalmente é feito com a adição de cloro, porém este produto apresenta algumas desvantagens em sua aplicação. Como alternativa ao cloro, algumas indústrias no Brasil e no exterior estão começando a utilizar outros biocidas, dentre estes o peróxido de hidrogênio, um poderoso oxidante que apresenta forte ação biocida. O objetivo deste trabalho foi comparar a eficiência do cloro e do peróxido de hidrogênio como biocidas em diferentes condições, através de testes em água da torre de resfriamento de uma indústria siderúrgica localizada no Rio de Janeiro. A contaminação microbiológica desta água foi medida sem a adição dos biocidas e com a adição de cloro e peróxido de hidrogênio, permitindo assim comparar o desempenho destas substâncias no combate aos grupos bacterianos presentes na amostra. Foi realizado também um estudo sobre o efeito corrosivo destas substâncias através de testes de corrosão em aço carbono 1020, que permitiram avaliar a taxa de corrosão por perda de massa provocada pela aplicação destes produtos na água. Os resultados mostraram que o peróxido de hidrogênio possui uma ação biocida satisfatória para aplicações em águas de resfriamento. Foi constatado que o efeito biocida do peróxido de hidrogênio é mais limitado do que o cloro e que sua eficiência depende do tempo de contato e pode ser afetada pela presença de impurezas dissolvidas na água. Os ensaios de corrosão revelaram que o peróxido de hidrogênio provoca um efeito corrosivo comparável ao do cloro no material testado. / [en] Cooling water treatment generally is made with the addition of chlorine, although it´s application has some disadvantages. There is an active development in Brazil and other countries to use alternative chemical disinfectants in place of chlorine, such as hydrogen peroxide, a powerful oxidant which is known for its high biocidal efficiency. The aim of this research is to study the effectiveness of hydrogen peroxide as a disinfectant compared to chlorine in different operational conditions. The experiments were carried out using an water sample from a cooling water system of a steelmaking plant in the city of Rio de Janeiro. The microbial contamination of this water sample was measured without adding any kind of disinfectant. After that, water sample was treated by adding hydrogen peroxide and chlorine, in order to compare and evaluate the efficiency of the two biocides to control bacterial growth in water. Besides microbiological tests, experiments were conducted to compare the degree of corrosion caused by the addition of hydrogen peroxide and chlorine in water. The experimental methodology employed 1020 carbon steel specimens and corrosion rates were measured by weight loss determination after the period of exposure. The results showed that the application of hydrogen peroxide leads to satisfactory bacterial control. However, compared to chlorine, hydrogen peroxide is a rather poor disinfectant. The efficiency of hydrogen peroxide depends on reaction time and it is affected by dissolved polluants in water. Evaluation of corrosion rates showed that hydrogen peroxide causes basically the same corrosion rates than chlorine.

Este estudo teve como objetivos: 01.Avaliar ex vivo o efeito de diferentes sistemas clareadores na microdureza dentinária em dentes bovinos submetidos ao clareamento dental interno. 02.Avaliar ex vivo o efeito de diferentes sistemas clareadores na morfologia superficial da dentina bovina. Fragmentos de 4 x 4mm, contendo esmalte e dentina, foram obtidos de coroas de incisivos bovinos extraídos. Os espécimes foram submetidos ao clareamento dental interno com peróxido de hidrogênio a 35% e peróxido de carbamida a 37% utilizando sistemas convencionais (Opalescence Endo® and Whiteness Super Endo®) e fotoativados (Opalescence Xtra® and Whiteness HP Maxx®). Os controles foram tratados com perborato de sódio misturado com peróxido de hidrogênio a 10% ou nenhum tratamento foi realizado. A microdureza dentinária foi mensurada antes e após os tratamentos clareadores, e os valores de dureza Knoop (KHN) foram submetidos à análise estatística (two-way ANOVA, Tukey\'s port-test). Os espécimes foram observados e fotografados sob microscópio eletrônico de varredura e avaliados com relação às alterações morfológicas da superfície da dentina. Houve uma redução significante na microdureza dentinária para todos os grupos testados quando comparados aos grupos controles. Opalescence Xtra® (-11.36 ± 8.14 KHN), Opalescence Endo® (-13.71 ± 8.02 KHN), Whiteness HP Maxx® (-15.18 ± 9.58 KHN) e Whiteness Super Endo® (-16.97 ± 6.55 KHN) foram semelhantes estatisticamente. O grupo do perborato de sódio misturado com peróxido de hidrogênio 10% (2.10 ± 8.58 KHN) e o grupo sem tratamento clareador (-2.71 ± 2.40 KHN) também foram estatisticamente semelhantes entre si. Ocorreu uma grande variação no padrão de alterações da morfologia superficial da dentina com os sistemas clareadores utilizados. Todos os produtos testados apresentaram redução significativa da microdureza dentinária, exceto o grupo de perborato de sódio misturado com peróxido de hidrogênio a 10%. Ambos, pH e oxidação do peróxido de hidrogênio apresentam o papel de alterar a estrutura da dentina durante o clareamento interno. A utilização de produtos alcalinos com um tempo reduzido de aplicação (técnicas fotoativadas) pode diminuir as alterações morfológicas na dentina. / The aim of this study was: 01. To evaluate in vitro the effect of different in-office bleaching systems on the surface morphology of bovine dentin. 02. To evaluate ex vivo the effect of different bleaching systems on the microhardness of bovine dentine. Tooth fragments measuring 4 x 4mm, containing enamel and dentin, were obtained from the crowns of extracted bovine incisors. Samples were submitted to simulated intracoronal bleaching techniques with 35% hydrogen peroxide and 37% carbamide peroxide using conventional (Opalescence Endo® and Whiteness Super Endo®) and light activated systems (Opalescence Xtra® and Whiteness HP Maxx®). Controls were treated either with sodium perborate mixed with 10% hydrogen peroxide or no bleaching agent. Dentine microhardness values were measured before and after bleaching procedures, recorded as KHN (Knoop Hardness Number), and the differences between them analyzed (two-way ANOVA, Tukey\'s port-test). The samples were observed under SEM and the recorded images were evaluated for topographic alterations. Significant reductions of dentine microhardness were observed for all treatments when compared to the control groups. Opalescence Xtra® (-11.36 ± 8.14 KHN), Opalescence Endo® (-13.71 ± 8.02 KHN), Whiteness HP Maxx® (-15.18 ± 9.58 KHN) and Whiteness Super Endo® (- 16.97 ± 6.55 KHN) presented similar differences. The walking bleach technique (2.10 ± 8.58 KHN) and the untreated groups (-2.71 ± 2.40 KHN) were statistically alike.The ultrastructural alterations of dentin observed in this study varied greatly between groups, according to the products used. Apparently, higher pH products associated to in-office techniques yielded to better maintenance of dentin ultrastructure. The in-office products tested in the present study caused a significant reduction in dentine microhardness. The walking bleach technique did not affect dentine microhardness. Both low pH and hydrogen peroxide oxidation play a role in altering the ultrastructure of dentin during internal dental bleaching. The use of alkaline products with reduced time of application (in-office techniques) may decrease such morphological alterations.

Danos induzidos por hiperglicemia em tecidos no diabetes são caracterizados por quatro mecanismos conectados: aumento do fluxo metabólico através da via do poliol, ativação da proteína quinase C (PKC), aumento da atividade da via das hexosaminas e aumento da produção intracelular dos precursores dos produtos finais de glicação avançada (AGEs). Entre eles, os derivados de metilglioxal, um potente agente de modificação de proteínas e DNA, têm sido associados a complicações microvasculares no diabetes: nefropatia, retinopatia e neuropatia. O metilglioxal é produzido a partir das trioses fosfato, acetona e aminoacetona, um catabólito de treonina e glicina, gerado na matriz mitocondrial. A aminoacetona sofre oxidação enzimática, catalisada por aminoxidase sensível a semicarbazida (SSAO), ou química, catalisada por íons de cobre e ferro, produzindo metilglioxal, H2O2 e NH4 +. Sabendo que metilglioxal e H2O2 são capazes de induzir apoptose e/ou necrose em células produtoras de insulina (RINm5f) propomos uma possível atividade pró-oxidante da aminoacetona sobre células beta do pâncreas. O tratamento destas linhagens com aminoacetona/Cu(II) aumentou a morte celular, fluxo de Ca2+ intracelular, produção de NO, fragmentação do DNA, depleção dos níveis de glutationa reduzida (GSH), expressão gênica da proteína apoptótica Bax, enzimas antioxidantes - glutationa peroxidase (GPx), glutationa redutase (GRd), catalase e isoformas de superóxido dismutases (CuZnSOD e MnSOD) - e óxido nítrico sintase induzida (iNOS). Embora as concentrações normais e patológicas da aminoacetona, provavelmente seja muito menores que as usadas nos experimentos, sugerimos que, em tecidos de diabéticos, um acúmulo da aminoacetona em longo prazo pode conduzir a danos oxidativos e eventualmente morte das células beta do pâncreas / Tissue damages induced by hyperglycemia in diabetics are characterized by four linked mechanisms: increased flux through the polyol pathway, protein kinase C (PKC) activation, increased hexosamine pathway activity and intracellular production of advanced glycation end product (AGE) precursors. The production of AGEs by modifying proteins and DNA agent, such as methylglyoxal, has been implicated in microvascular complications in diabetes: nephropathy, retinopathy and neuropathy. Methylglyoxal is putatively produced in vivo from trioses phosphate, acetone and aminoacetone, a catabolite of threonine and glycine synthesized in the mitochondrial matrix. Aminoacetone has been reported to undergo semicarbazide sensitive amine oxidase- catalyzed and copper- and iron-catalyzed oxidations by molecular oxygen to methylglyoxal, NH4 + ion and H2O2. Considering that methylglyoxal and H2O2 have been found to promote apoptosis/necrosis to insulin-producing cells (RINm5f), we propose a possible pro-oxidant role of aminoacetone in pancreatic beta-cells. Treatment of RINm5f cells with aminoacetone plus Cu(II) ion promotes an increase of non-viable cells, influx of Ca2+ ions, NO production, DNA fragmentation, depletion of reduced glutathione (GSH) levels, and increased mRNA expression of pro-apoptotic protein (Bax), antioxidant enzymes - glutathione peroxidase (GPx), glutathione reductase (GRd), MnSOD, CuZnSOD and catalase - and inducible nitric oxide synthase (iNOS). Although both normal and pathological concentrations of aminoacetone are probably much lower than those used here, it is tempting to propose that excess aminoacetone in diabetic patients, at long term, may drive oxidative damage and eventually death of pancreatic beta-cells

Este trabalho foi dividido em quatro etapas, além da introdução. A primeira parte consiste no estudo comparativo do comportamento eletroquímico de eletrodos de cobre em soluções tampão fosfato 0,10 mol L-1 com valores de pH 4,5 e 7,8, e em solução de NaOH com valores de pH 13,0 e 14,0. Mostrou-se que as espécies formadas eletroquimicamente sobre a superfície do eletrodo são dependentes do valor de pH e do potencial aplicado. Os resultados experimentais obtidos estão de acordo com aqueles reportados na literatura. A segunda parte da tese corresponde ao desenvolvimento de um sensor eletroquímico fabricado com microeletrodos de cobre para a determinação de peróxido de hidrogênio (H2O2) em amostras comerciais de antissépticos bucais e clareadores dentais. Nas condições experimentais otimizadas (solução tampão fosfato 0,10 mol L-1 (pH 7,0) e potencial de redução de -0,20 V), microeletrodos de cobre foram utilizados para a determinação de H2O2 sem a necessidade de etapas de extração, obtendo-se faixa linear de 0,015 a 6,4 mmol L-1 e limite de detecção de 2,8 &#181;mol L-1. O método proposto mostrou-se reprodutível e a presença de interferentes na matriz da amostra analisada não alterou a resposta do sensor para H2O2. Na terceira parte, a reação de eletro-oxidação de glicerol em superfícies de cobre em soluções de NaOH foi investigada utilizando a microscopia eletroquímica de varredura (SECM) no modo de geração pelo substrato de cobre / coleta no microeletrodo de platina (tip) (SG/TC). Os experimentos com a SECM mostraram a dependência da corrente com a distância entre o gerador e a tip, assim como a dependência com a concentração da solução de NaOH. Além disso, a corrente monitorada no microeletrodo diminuiu significativamente após a adição de glicerol. A atividade eletrocatalítica de eletrodos de cobre, no que diz respeito à oxidação de glicerol em meio alcalino, permitiu o desenvolvimento de um sensor amperométrico para a determinação de glicerol em amostra de biodiesel, com um intervalo linear de 0,05 a 1,33 mmol L-1 e limite de detecção de 20 &#181;mol L-1. A metodologia proposta foi aplicada para a determinação do analito em uma amostra de biodiesel de mamona. A quarta parte descreve a síntese, caracterização eletroquímica e utilização de nanotubos de carbono de paredes múltiplas (NCPM) decorados com nanopartículas de paládio (Pd) para a modificação da superfície de eletrodo de carbono vítreo (CV) para a oxidação de metanol, etanol e glicerol em solução de KOH 1,0 mol L-1. Os resultados mostraram que o NCPM/Pd é um bom catalisador para a eletro-oxidação de álcoois em meio alcalino e que maior atividade eletrocatalítica foi obtida para o glicerol. Em condições otimizadas, experimentos amperométricos foram realizados para o desenvolvimento do método analítico para a determinação de glicerol em biodiesel, obtendo-se um intervalo linear de 0,06 a 24 mmol L-1 e limite de detecção 30 &#181;mol L-1. Amostras de biodiesel foram analisadas com sucesso por meio de curvas de adição de padrão utilizando o eletrodo CV/NCPM/Pd. / This thesis is divided into four parts, apart from a general introduction. Part 1 is concerned with a critical comparison of the electrochemical behaviour of copper electrodes, in 0.10 mol L-1 phosphate buffer solution (pH 4.5 and 7.8), and in NaOH solution (pH 13.0 and 14.0). The electrochemical study showed that the species formed on the surface depends on both pH and the applied potential. The experimental results are in good agreement with those reported in the literature. A second goal of the thesis was the development of an electrochemical sensor for hydrogen peroxide (H2O2) determination in commercial oral antiseptic and dental whitening samples using copper microelectrodes. With optimized experimental conditions (working potential applied = -0.20 V in 0.10 mol L-1 phosphate buffer solution pH= 7.0), copper microelectrodes can be used to detect H2O2 without any previous extraction steps, in the range of 0.015 to 6.4 mmol L-1 with a lower detection limit of 2.8 &#181;mol L-1. In addition, the electrode exhibited an excellent reproducibility and long-term stability as well as negligible interference from sorbitol, ethanol, glycerin and saccharin. In the third part, the electrooxidation reaction of glycerol at copper surfaces in NaOH solutions was investigated using Scanning Electrochemical Microscopy (SECM) in a copper substrate generation / platinum microelectrode (tip) collection mode (SG/TC). The results showed the dependence of the current measured at the tip with respect to the distance between generator and the tip, as well as on the concentration of the NaOH solution. Other studies led to the conclusion that the current measured at the tip decreased significantly after addition of glycerol in the solution. The electrocatalytic activity of copper electrodes for the oxidation of glycerol in alkaline medium allowed the development of an amperometric method to determine glycerol in biodiesel samples in the range 0.05 to 1.33 mmol L-1 with a detection limit of 20 &#181;mol L-1. The determination of the analyte in a castor biodiesel sample employing a single copper microelectrode was carried out. The four part of this thesis shows results on the synthesis, electrochemical characterization and use of a glassy carbon (GC) electrode modified with multi-wall carbon nanotubes (MWCNT) decorated with palladium (Pd) for the electrochemical oxidation of methanol, ethanol and glycerol in 1.0 mol L-1 KOH solution. The results showed that MWCNT/Pd is a good electrocatalyst for methanol, ethanol and glycerol oxidation in alkaline medium, with highest activity toward glycerol oxidation. With optimized experimental conditions, an amperometric method was developed for the determination of glycerol in biodiesel samples, in the range of 0.06 to 24 mmol L-1 and detection limit of 30 &#181;mol L-1. Biodiesel samples were successfully analyzed by the standard addition method using the GC/MWCNT/Pd film electrode.

Em âmbito hospitalar é crescente o emprego de dispositivos confeccionados de distintos materiais termossensíveis. Assim, o emprego de metodologias esterilizantes compatíveis tem sido o foco de muitas pesquisas, dentre as quais destacam-se estudos envolvendo o plasma. O mecanismo de ação deste desenvolve-se com a aplicação de Rádio-Freqüência a gases precursores, resultando na inativação microbiana por espécies altamente reativas. Este método inovador caracteriza-se por não gerar riscos de toxicidade ocupacional e aos pacientes, e ser processado em temperatura próxima ao ambiente. Para análise comparativa foi utilizado o método de esterilização por óxido de etileno (agente químico na forma gasosa). Este gás apresenta características de elevada inflamabilidade, explosividade e toxicidade, por isso é usado diluído em gases inertes, além de deixar residual no material esterilizado, tendo que ter um controle rigoroso no processo de aeração; porém atualmente é um dos métodos mais utilizados para esterilização de materiais odonto-médico-hospitalares, particularmente os termossensíveis. O principal objetivo deste trabalho foi estudar diferentes parâmetros do processo e seus respectivos resultados, que influenciam na esterilização empregando plasma e compará-los com os obtidos empregando óxido de etileno. O equipamento utilizado para o estudo dos processos de esterilização por plasma foi o ICP (Inductively Coupled Plasma). Analisou-se assim para o plasma algumas distintas combinações de parâmetros, tais como: gases (oxigênio puro e mistura deste com peróxido de hidrogênio a 5,10 e 20%), pressão (330 mTor), vazão do gás (100sccm), temperatura (próxima ao ambiente), potência de rádio-freqüência (300, 350 e 400W), tempos de exposição (com intervalos de 3 a 60 min) e umidade relativa (80±5%). No ICP foram desenvolvidas duas fases planejadas para os processos, seguindo uma programação experimental, já no óxido de etileno foram realizadas três séries de exposições sub–letais utilizando mistura esterilizante Oxyfume® 2002 (10% Óxido de Etileno, 63% HCFC 124 e 27% HCFC 22), sendo os parâmetros padronizados: umidade relativa (40 a 60%), concentração do gás (450 mg/L), temperatura (55° C) e tempos de exposição (com intervalos de 3 a 15 min.). Todos os ciclos foram realizados em triplicata. Esporos de Bacillus subtilis var. niger ATCC 9372 foram obtidos a partir de suspensões de microrganismos e inoculados em suportes na concentração de 107 UFC/suporte para serem utilizados nos estudos dos processos de esterilização. Empregou-se a técnica de Pour Plate (incubação em estufa por 24hs a 37 °C) para a quantificação dos esporos. Para o processo de esterilização por plasma os resultados obtidos forneceram valores D que variaram entre 8 e 3 min., dependendo dos parâmetros testados, e para o processo de esterilização por óxido de etileno o Valor D foi de 2,80 min. Concluiu-se que o processo de esterilização por plasma apresentou resultados interessantes e promissores e os melhores resultados foram obtidos com as potências maiores de 350 e 400W para o gás oxigênio puro, caracterizando o plasma como alternativa promissora de esterilização, devido às suas características positivas frente ao óxido de etileno, pois os valores D entre os dois processos de esterilização não apresentaram uma diferença significativa. / The gas Plasma sterilization technology has been emerging as an alternative to conventional low temperature processes since the advent of new therapies using heat sensitive materials in the healthcare field is greater than ever. The gas Plasma mechanism of action includes the generation of actives species by an electrical discharge, which is able to promote lethal effect on microorganisms. The sterilization techniques using gas plasma are under intense investigation and it has already been demonstrated by recent studies that this technology is simple, cost-effective, suitable for microbicidal activity and absent of toxic residuals. Ethylene oxide is the sterilization agent most widely applied to medical devices. However, its explosiveness, inflammability and toxicity led to the search for other sterilization methods at low temperature and it has been used associated to non active gases which inhibit these properties and it is necessary to have the control at the occupational safety and environmental monitoration. Therefore, the aim of this work was to explore possible microbicidal application of the gas plasma sterilization generated by an inductively coupled system and to compare this sterilization method with ethylene oxide (chemistry substance in gaseous form), observing their D value. It was used distinct combinations of process parameters to sterilization by plasma, as follows: radio-frequency powers (300, 350 and 400 watts), exposition times (in the range of 3 to 60 minutes), gas (pure oxygen and mixture with hydrogen peroxide 5,10 and 20%), gas flow (100 sccm), pressure (330 mTorr), temperature (close to the environmental one) and relative humidity 80±5%. For ethylene oxide, Oxyfume 2002® was used (mixture of ethylene oxide, HCFCs 22 and 124), under the concentration of 450mg/L, at the temperature of 55°C and relative humidity of 50±5% and it was submitted to a time of exposition between 3 to 15 minutes. Both processes were submitted to exposition cycles in triple. Bacillus subtilis var. niger ATCC 9372 inoculated in a standard load of 107 spores per carrier was used as biological indicator. After the exposition, the biological indicator\'s spore survivors were counted by the \"Pour Plate\" technique (incubation temperature of 35 ± 2ºC for 24 hours). Significant microbial reduction was observed in some cases where the plasma D value was between 3 and 8 min and 3,08 min, 3,04 min. to 350 and 400W powers respectively. In the ethylene oxide process the D value was 2,80 min. These results evidenced the higher effectiveness of ethylene oxide comparatively to plasma. However the latter presents advantages that make it an interesting alternative to low temperature sterilization processes.